Article
pubs.acs.org/biochemistry
Effects of Macromolecular Crowding on Alcohol Dehydrogenase
Activity Are Substrate-Dependent
A. E. Wilcox, Micaela A. LoConte, and Kristin M. Slade*
Department of Chemistry, Hobart and William Smith Colleges, Geneva, New York 14456, United States
ABSTRACT: Enzymes operate in a densely packed cellular environment that rarely matches the dilute conditions under which
they are studied. To better understand the ramifications of this crowding, the Michaelis−Menten kinetics of yeast alcohol
dehydrogenase (YADH) were monitored spectrophotometrically in the presence of high concentrations of dextran. Crowding
decreased the maximal rate of the reaction by 40% for assays with ethanol, the primary substrate of YADH. This observation was
attributed to slowed release of the reduced β-nicotinamide adenine dinucleotide product, which is rate-limiting. In contrast, when
larger alcohols were used as the YADH substrate, the rate-limiting step becomes hydride transfer and crowding instead increased
the maximal rate of the reaction by 20−40%. This work reveals the importance of considering enzyme mechanism when
evaluating the ways in which crowding can alter kinetics.
C
enhancing product inhibition,21 changing solution polarity,18 or
slowing diffusion10,22,23 and thus reducing the frequency of
substrate−enzyme encounters.12,24,25 At the same time,
enhancements in enzyme activity can occur due to conformational changes,26−29 restricted internal dynamics,20 or changes
in oligomerization state.30 Because the relative influence of
these factors is not well understood, crowding trends for
enzyme kinetics are difficult to predict. Studies have begun to
investigate how enzyme size and crowding agent properties
determine the dominant factors in crowding effects,31−33 yet to
the best of our knowledge, no one has directly examined the
role of enzyme mechanism. Fortunately, yeast alcohol
dehydrogenase (YADH) provides a unique opportunity to do
so because the rate-limiting step of the mechanism depends on
its substrate.
YADH is a 150 kDa tetramer that catalyzes the reversible
oxidation of an alcohol (S) to an aldehyde or ketone (P) using
the cofactor NAD+ as illustrated in Scheme 1. The reaction
consists of a stereospecific hydride transfer from the alcohol to
the cofactor and a series of conformational changes that are
likely to be influenced by crowding.34 For the natural substrate
ethanol (EtOH), the slow step of the mechanism is the release
ellular concentrations of proteins, nucleic acids, and
polysaccharides are tens to hundreds of times greater than
those employed for the dilute conditions used in most
biophysical studies.1−3 To better mimic and understand the
ramifications of this densely packed environment, high
concentrations of polymers such as dextran, ficoll, or
polyethylene glycol are often added to in vitro conditions.
These experiments, in combination with theoretical and
computational work, have revealed that macromolecular
crowding significantly alters protein dynamics, diffusion, and
chemical equilibria, as well as other biophysical properties.4−6
Recent experiments have applied these techniques in
combination with Michaelis−Menten kinetics to better understand how crowding affects enzyme activity.7,8
In general, crowding has little to no effect on the Michaelis
constant, Km, of enzymes.9 Several studies with diffusion-limited
enzymes report small increases in Km due to diffusion
resistance.10−12 In the overwhelming majority of cases,
however, the presence of crowders results in slight decreases
in Km.9,13−16 This reduction is frequently attributed to changes
in activity coefficients and increased effective concentrations
that can enhance substrate binding.17−20
In contrast to Km, the effects of crowding on Vmax, the
maximal velocity, are more varied and appear to depend on
numerous, sometimes competing factors. For example,
crowding has been reported to decrease enzyme activity by
© 2016 American Chemical Society
Received: March 21, 2016
Revised: June 8, 2016
Published: June 9, 2016
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Scheme 1
of the NADH product.35 For larger alcohols, such as
isopropanol and benzyl alcohol, the hydride transfer becomes
rate-limiting.36−38 Thus, this system can provide further insight
into whether the rate-limiting step (RLS) of a single enzyme
plays a role in determining how crowding influences enzyme
kinetics.
v0 =
EXPERIMENTAL PROCEDURES
Chemicals. Isopropanol, isopropanol-d8, β-nicotinamide
adenine dinucleotide hydrate (NAD+), bovine serum albumin
(BSA) as purified lyophilized powder, alcohol dehydrogenase
(EC 1.1.1.1) from Saccharomyces cerevisiae (YADH, 361 units/
mg) as a lyophilized powder, and dextran polymers from
Leuconostoc mesenteroides (∼9−11, 150, and 200 kDa) and
Leuconostoc spp. (∼40 and 450−650 kDa) were obtained from
Sigma-Aldrich. Dextran polymers from L. mesenteroides were
also purchased from Molecular Probes (∼86 kDa). βNicotinamide adenine dinucleotide disodium salt trihydrate,
in its reduced form (NADH), was purchased from Amresco.
Absolute anhydrous ethanol was purchased from PharmcoAaper, and anhydrous ethanol-d6 was from Cambridge Isotope
Laboratories. Glucose and sodium pyrophosphate were from
Acros Organics. Solutions were prepared with 100 mM
pyrophosphate buffer (pH 8.9), and the pH of crowding
agent solutions was corrected to 8.9 before their use.
Michaelis−Menten Kinetic Assays. YADH activity at 25
°C was determined by monitoring the change in NADH
absorbance at 340 nm every 6 s for 6 min in a 96-well plate with
a Molecular Devices SpectraMax 190 spectrophotometer while
the plate was being shaken. Each well contained 110 mM
ethanol or ethanol-d6, 0.125−4.00 mM NAD+, 0.024 unit/mL
(0.45 nM) YADH, and 300 g/L crowding agent. The YADH
stock solution was prepared by adding 8 μL of a 2 g/L enzyme
solution to 5992 μL of 1 g/L BSA, which served to stabilize and
maintain enzyme activity.39 Addition of ethanol initiated the
enzymatic reaction, but adding the reagents in a different order
did not have an effect on the resulting rate. The assay was
repeated using 550 mM isopropanol or isopropanol-d8 in place
of ethanol and 0.25−12.5 mM NAD+. The isopropanol stock
solution was kept warm throughout the experiment to prevent
precipitation. Addition of YADH initiated the reaction. In the
absence of YADH or one of the substrates, the absorbance in
the well did not change with time, indicating no detectable
enzyme activity. For a given set of conditions, an assay was
repeated eight times.
Michaelis−Menten Data Analysis. Because saturating
alcohol concentrations were employed, the YADH-catalyzed
oxidation of ethanol or isopropanol by NAD+ can be treated as
single-substrate reactions with the following scheme:
k1
k −1
(2)
Initial enzymatic rates, vo, were obtained by taking the maximal
slopes from absorbance versus time plots using SoftMax Pro
version 6.3. A single Michaelis−Menten curve was then
constructed by averaging eight initial rates per substrate
concentration [S], for eight concentrations, using SigmaPlot
to determine Km and Vmax. Km and Vmax values obtained under
crowded conditions were normalized to the values obtained in
buffer only, yielding relative kinetic values.
Benzyl Alcohol YADH Kinetic Assays. The reaction
progress of YADH was followed using a 96-well plate Biotek
Eon spectrophotometer. Each well contained 75 mM benzyl
alcohol or ethanol, 1.5 mM NAD+, and 12 units/mL (0.225
μM) YADH in the presence of 300 g/L glucose or dextran.
Concentrations of all substrates were chosen to be saturating,
and 100 mM pyrophosphate buffer (pH 8.9) was used to bring
the final volume to 200 μL. All solutions were kept on ice to
preserve their integrity. The YADH stock was prepared by
adding 1.2 mL of a 1 g/L solution of YADH to 1.8 mL of 1 g/L
BSA, which served to stabilize and maintain enzyme activity.
Immediately following the addition of 10 μL of the YADH
stock to the assay, the rate of NADH formation was measured
at 340 nm every 40 s for 6 min. All assays were repeated 18
times. Initial enzymatic rates were obtained by taking the
maximal five-point slopes from absorbance versus time plots
using Gen5 software. These initial rates recorded in the
presence of glucose or dextran were then normalized to the
initial rates obtained in buffer only, yielding relative rates.
Fluorescence Studies. All fluorescence experiments were
performed at 25 °C using a PerkinElmer LS 45 fluorimeter with
excitation and emission slits set to 10 nm. A 0.09 μM YADH
solution was titrated with glucose, 150 kDa dextran, or buffer.
To monitor the resulting tryptophan fluorescence, an excitation
wavelength of 295 nm was used to record emission spectra
from 300 to 400 nm. Fluorescence intensities from both
titrations were corrected for dilution.
Coenzyme Binding. Because coenzyme binding quenches
YADH fluorescence, an excitation wavelength of 295 nm and
an emission wavelength of 340 nm were used to monitor
tryptophan fluorescence. Titration experiments were performed
by adding small aliquots of 75.4 mM NAD+ or 2.11 mM
NADH to 3.0 mL of 0.90 μM YADH in 100 mM
pyrophosphate buffer (pH 8.9) in the presence or absence of
300 g/L glucose. Data were analyzed as previously described.42
In short, background readings were subtracted and corrections
made to compensate for enzyme dilution. The fractional
saturation of coenzyme, θ, was determined from
■
θ=
kcat
E + S HooI ES ⎯→
⎯ E+P
Vmax[S]
K m + [S]
(1)
F° − F
F ° − F′
(3)
where F° is the fluorescence intensity in the absence of
coenzyme, F′ is the fluorescence intensity at saturating
concentrations, and F is the intensity after addition of a given
concentration of coenzyme, [C]. SigmaPlot software was used
to determine the dissociation constant, KD, from
where E is the enzyme, S is the substrate, and k1, k−1, and kcat
are rate constants as first described by Brown.40 The maximal
rate, Vmax, and the Michaelis constant, Km, can then be
determined from the Michaelis−Menten equation:41
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Figure 1. Concentration-dependent effects of dextran on YADH Michaelis−Menten plots. Assays containing YADH, (A) ethanol or (B)
isopropanol, and varying concentrations of NAD+ were performed in the presence of 0 (black), 25 (green), 100 (purple), 200 (blue), or 300 g/L
(red) 150 kDa dextran in 100 mM pyrophosphate buffer (pH 8.9). Error bars represent standard deviations (n = 8).
Figure 2. Effects of crowding on YADH kinetic parameters depend on the substrate and are independent of dextran size. Assays containing YADH,
ethanol (black) or isopropanol (red), and varying concentrations of NAD+ were performed in the presence of 300 g/L glucose or various sizes of
dextran (kilodaltons). (A and C) Vmax and (B and D) Km values from the resulting eight-point Michaelis−Menten curves were normalized to values
acquired in the absence of dextran or glucose to yield the relative kinetic constants shown above (y-axes). Error bars represent ± one standard error
(n = 8). Asterisks (*p < 0.0003; **p < 0.06; ***p < 0.1) indicate a significant difference in the relative Vmax of (A) ethanol vs isopropanol or (C)
dextran vs glucose (Student’s two-tailed t test). For ethanol, Student’s two-tailed t tests failed to show any statistical difference between the relative
(C) Vmax (all p values of >0.1) or (D) Km (all p values of >0.5) for any two sizes of dextran.
θ=
[C]
KD + [C]
(4)
■
RESULTS
The initial rates for the YADH-catalyzed oxidation of alcohol
were monitored spectroscopically for varying concentrations of
NAD+. The resulting Michaelis−Menten curves (Figure 1)
provided Km and Vmax parameters. To account for day-to-day
variability in the assays, relative values were determined by
dividing the Km and Vmax parameters obtained in glucose or
dextran by values obtained concurrently in the absence of these
sugars.
The results indicate that the effects of crowding depend on
the alcohol used as the YADH substrate. While the presence of
glucose or dextran decreases the rate of YADH activity for
ethanol oxidation, these sugars tend to enhance YADH activity
for isopropanol oxidation (Figure 2A). Similar rate enhancements were also observed when benzyl alcohol was used as the
YADH substrate (Figure 3). The relative Km values for NAD+
are consistently larger when isopropanol is used as the substrate
Figure 3. Effects of crowding on YADH activity are altered when
benzyl alcohol is substituted for ethanol substrate. YADH assays
containing saturating concentrations of either ethanol (black) or
benzyl alcohol (red) and NAD+ were monitored in the presence of
300 g/L glucose or 150 kDa dextran. These initial rates were divided
by initial rates obtained in buffer only to yield relative rates (y-axis).
Error bars represent ± one standard error (n = 18). To compare the
ethanol and benzyl alcohol rates, p values (two-tailed) were calculated
for t tests assuming equal variance.
in place of ethanol (Figure 2B). While Figure 2B is suggestive
of a potential dextran size dependence for ethanol oxidation,
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testing a wider range of dextran dimensions reveals that
crowding effects on both Vmax and Km are independent of
polymer size (Figure 2 C,D).
To confirm that the opposing crowding effects with ethanol
and isopropanol are due to different rate-limiting steps of the
enzyme, the YADH assays were repeated with deuterated
substrates (Figure 4). No difference in relative Vmax values
Figure 5. Glucose has a negligible effect on binding of NAD+ to
YADH. Binding was monitored by a decrease in tryptophan
fluorescence intensity (λex = 295 nm; λem = 340 nm) as NAD+ was
titrated into a solution of 0.90 μM YADH in the presence (red) or
absence (black) of 300 g/L glucose. Fractional saturation of the
enzyme was determined as described in Experimental Procedures after
accounting for dilution. Error bars represent ± one standard error (n =
3).
Figure 4. Isotopic labeling of the alcohol substrate. Relative Vmax
values were obtained for the YADH-catalyzed reaction described in
Figure 2 in the presence and absence of 300 g/L 150 kDa dextran. The
substrate was either perdeuterated (gray, isopropanol-d8 or ethanol-d6)
or not (blue). Error bars represent ± one standard error (n = 8). To
compare the hydrogen and deuterium relative Vmax values, p values
(two-tailed) were calculated for two-sample t tests: equal variance was
assumed for ethanol, but not for isopropanol.
for NADH titrations with YADH in the presence of dextran
(Figure 6B).
On the basis of the drastic difference in YADH tryptophan
fluorescence in the presence and absence of dextran (Figure 6),
additional fluorescence experiments were conducted to
investigate the effects of this polymer on YADH. The combined
intensity decrease and red-shift in fluorescence with increasing
150 kDa dextran concentration indicate a change in the
microenvironment of the YADH tryptophan residues (Figure
7). In contrast, the YADH fluorescence is essentially unaltered
by the addition of glucose.
should be observed with different alcohol isotopes if crowding
primarily impedes NADH release. In contrast, if crowding
instead influences hydride transfer, different relative Vmax values
are likely, in part, because deuterium requires a donor−acceptor
distance (DAD) for the hydrogen tunneling process shorter
than that of hydrogen.46 For isopropanol, the data are
consistent with the latter, showing that the relative Vmax value
with hydrogen was only two-thirds the value of the
perdeuterated substrate. In contrast, the presence of dextran
decreased the YADH activity with ethanol by 40% regardless of
whether the alcohol was deuterated. The drastic increase in the
relative Vmax when deuterating isopropanol contrasted with the
lack of an effect for ethanol suggests that the implications of
crowding on YADH kinetics are related to its rate-limiting step.
The decrease in Km values observed with crowding is
frequently attributed to enhanced substrate−enzyme binding.17−20 To investigate this possibility, ligand binding curves
generated from fluorescence measurements were used to
determine the dissociation constant, KD, of NAD+ for YADH
(Figure 5). Because tryptophan fluorescence of YADH is
quenched by coenzyme binding, titrating this enzyme with
increasing concentrations of NAD+ resulted in a decrease in
fluorescence intensity (Figure 6). Under dilute conditions
(absence of glucose or dextran) with isopropanol as the
substrate, the resulting KD value was within error of the Km for
NAD+ (p value = 0.9, from a Student’s two-tailed t test), yet
when ethanol was the substrate, the NAD+ Km value was
statistically different (p value = 0.002, from a Student’s twotailed t test) from the corresponding KD value (Table 1).
Furthermore, the presence of glucose had little to no influence
on YADH−coenzyme binding with a relative KD for NAD+ of
0.91 ± 0.03. Corresponding values could not be obtained with
dextran solutions because the first addition of NAD+ to a
YADH solution containing dextran resulted in an increase in
fluorescence intensity, rather than the expected decrease
observed in buffer (Figure 6A). Similar effects were observed
■
DISCUSSION
The effects of dextran and glucose on the kinetics of alcohol
dehydrogenase depend on the alcohol employed in the assay.
This result is most likely due to the substrate-dependent
mechanism of YADH. Specifically, crowding decreases the rate
of ethanol oxidation, which is limited by product release. In
contrast, when YADH exhibits slow catalysis through the use of
isopropanol or benzyl alcohol as its substrate, crowding
enhances enzymatic activity (Figures 2A and 3). Moreover,
the concentration-dependent effects of dextran differ for
ethanol (Figure 1A) and isopropanol (Figure 1B). This
observation further supports our conclusion that the mechanism by which crowding alters YADH activity is different for
ethanol and isopropanol oxidation.
A single study previously called attention to the substratespecific effects of crowding, attributing the observation instead
to differences in the chemical interactions of each substrate
with the surrounding crowders.13 To verify that the substratespecific crowding effects observed in our study are due to
different rate-limiting steps of the enzyme and not differences
in the chemical properties of these substrates, the YADH assays
were repeated with deuterated alcohols. The rate of hydride
transfer during YADH catalysis is affected by the specific
isotope used in the alcohol substrate, but the release of NADH
from the active site is not. The fact that isotopic substitution of
the substrate altered the relative Vmax for isopropanol oxidation
but not ethanol oxidation (Figure 4) suggests that crowding
does not influence the same step of these two reactions even
though they are catalyzed by the same enzyme. Taken together
with Figures 2A and 3, this work provides the first evidence that
effects of crowding on enzyme kinetics depend on the ratelimiting step of the mechanism.
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Figure 6. Coenzyme binding of YADH. Tryptophan fluorescence (λex = 295 nm; λem = 340 nm) of 0.90 μM YADH was monitored with sequential
the additions of (A) NAD+ or (B) NADH in the presence (red) or absence (black) of 300 g/L 150 kDa dextran.
Table 1. Michaelis and Dissociation Constants for NAD+
with YADH in a Dilute Solution
Km (μM) with ethanol
Km (μM) with isopropanol
KD (μM)
270 ± 90
840 ± 40
850 ± 30
for ethanol oxidation in glucose with slowed small-molecule
diffusion.23 This observation is consistent with numerous
crowding reports that attribute decreased enzymatic activity to
the reduced frequency of substrate encounters.12,24,25 For
example, lactate dehydrogenase (LDH) operates very near or at
diffusion-limited rates,44 such that crowding slows its activity by
impeding substrate−enzyme encounters.15 However, this
explanation is inappropriate for YADH because ethanol
Vmax Analysis. Our previous work with YADH used
electrochemical experiments to correlate the decrease in Vmax
Figure 7. Effect of glucose and dextran on the tryptophan fluorescence of YADH. A 0.09 μM YADH solution was titrated with (A) glucose or (B)
150 kDa dextran. Using an excitation wavelength of 295 nm, the resulting emission spectra were corrected for dilution. (C) The 150 kDa dextran
elicited a detectable 0−14 nm red-shift in the emission wavelength of maximal fluorescence.
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caging.43 The presence of dextran has previously been shown
to augment inhibition effects, potentially by increasing the
effective concentration of the inhibitor.21 Furthermore, NAD+
inhibition of YADH has been well documented at high
coenzyme concentrations in the absence of crowding.50−53
Nonetheless, future experiments are necessary to determine if
substrate inhibition, slowed product release, or an additional
factor is responsible for the observed decrease in the extent of
isopropanol oxidation with large dextrans.
Km Analysis. The Michaelis constant is defined by the
equation Km = (k−1 + kcat)/k1 using the rate constants in eq 1.
For reactions in which the chemistry is slow, kcat is often small
relative to k−1, such that the Km can be approximated as the
dissociation constant (KD = k−1/k1). As a result of this
assumption, improved substrate binding has been widely used
to rationalize the observed decreases in Km with crowding.17−20
For YADH, however, this is not an appropriate explanation.
Even in dilute solutions, the KD value associated with NAD+ is
3-fold greater than the NAD+ Km value for ethanol oxidation
(Table 1), suggesting that Km reflects more than merely
coenzyme binding and that catalysis is not slow enough for kcat
to be negligible in this system. Furthermore, if Km was a
reflection of binding (Km ≈ KD), the Km values for NAD+
should be similar regardless of the alcohol used in the assay.
However, the Km for NAD+ in dilute solution increases by 3fold when ethanol is replaced with isopropanol (Table 1).
The observations mentioned above are consistent with our
knowledge of the YADH mechanism. It is not surprising that
kcat contributes significantly to Km for ethanol oxidation,
because the chemistry is not slow.56 In fact, a more complex
model derived by Northrop57 is helpful for analyzing this
system in which kcat is separated into a rate constant for
catalysis (k2) and release (k3):
oxidation is limited by NADH release. Thus, the decrease in
YADH activity is more likely to arise from glucose and dextran
impeding diffusion of the product from the active site. This
justification is indeed supported by the isopropanol and benzyl
alcohol data. When NADH release is not the slow step, both
glucose and dextran increase YADH activity instead.
Consistent with our previous work,23 the relative Vmax for
ethanol oxidation in the presence of glucose is lower than that
for dextrans (Figure 2). We previously proposed that the
presence of dextran adds an additional macromolecular
crowding effect that partially offsets the decreased activity
observed with glucose (from slowed product release). At the
time, however, no direct evidence of the specific source of this
effect was available. Figure 7 now provides more insight,
revealing that dextran affects the YADH structure in ways that
its small-molecule counterpart cannot. Specifically, the addition
of dextran to YADH solutions results in a red-shift and a
decrease in fluorescence intensity, indicating a change in the
tryptophan microenvironment. In contrast, the YADH
fluorescence is essentially unaltered by the addition of glucose,
suggesting the effect with dextran is not due to polarity or other
chemical effects. Similar trends in tryptophan fluorescence have
been observed for at least four other enzymes, suggesting that
crowding commonly induces structural changes in enzymes.45
This structural change most likely is the system’s way of
reducing excluded volume effects and compensating for the
additional entropy pressure imposed by crowding.
In contrast, both dextran and glucose have similar effects on
isopropanol oxidation, suggesting that the observed rate
enhancement is more likely a result of chemical effects than
crowding (Figure 2A). Because the rate of the hydride transfer,
the slowest step for this mechanism,38 depends on the distance
between the donor and the acceptor within the active site,46,47
these sugars likely compact the YADH globule, which
subsequently optimizes the transfer distance. This claim is
supported by a study analyzing the effects of osmolytes on the
rate constants and structure of lactate dehydrogenase (LDH).48
Specifically, TMAO stabilizes LDH by compacting it into a
protein globule, which enhances the hydride transfer crucial for
catalysis and decreases the activation energy by 15-fold. While
TMAO is chemically distinct from glucose, it exhibits chemical
properties comparable to those of the osmolyte betaine, which
has been used to study YADH. In fact, betaine and glucose have
similar effects on YADH stabilization.49 Thus, it is likely that
the effects of glucose on YADH compaction parallel those
observed with LDH in the presence of TMAO. It then follows
that the rate enhancement observed with glucose and dextran
results from stabilization and compaction of YADH, which
streamlines the efficiency of the hydride transfer.
The one exception to the dextran rate enhancement of
isopropanol oxidation occurred in the presence of the largest
dextran size (Figure 1A, red). At the concentrations of dextran
employed in our experiments, theory suggests that polymer
entanglement creates a depletion layer that can cage YADH
together with substrates and products.54 This caging could
further impede NADH release. Because the depletion layer
becomes thicker with increasing polymer size,55 the largest
dextran is presumably the most effective at caging. Consequently, at the largest dextran size, the effect from caging and
slowed product release may dominate over the competing effect
of the crowding-enhanced hydride transfer. Alternatively, the
observed reduction in YADH activity for 450−550 kDa dextran
could be a result of enhanced substrate inhibition from
k1
k2
k3
E + S HooI ES → EP → E + P
k −1
(5)
where ethanol is in excess, E is the YADH enzyme, S is NAD+,
P is NADH, and Km is defined by
Km =
k 3(k −1 + k 2)
k1(k 2 + k 3)
(6)
The Michaelis constant for this system can be further
simplified to Km ≈ k3/k1,58 because product release is slow59 (k2
≫ k3), and Leskovac et al.56 showed that catalysis is faster than
release (k2 ≫ k−1). Consequently, the decrease in Km observed
in the presence of glucose or dextran (Figure 2B) is most likely
due to impeded product release, k3. This argument is also
supported by the fact that crowding has similar effects on the
NAD+ Km values (Figure 2B) and the ethanol Km values
previously reported for the same system,23 which would not
necessarily be the case if Km were a reflection of substrate
binding.
In contrast to ethanol oxidation, the NAD+ Km value more
closely corresponds its KD value for isopropanol (Table 1). For
larger alcohol substrates, like isopropanol and benzyl alcohol,
the hydride transfer that occurs during YADH catalysis
becomes limiting,36−38 such that kcat may be small enough
relative to k−1 for the Km to begin approximating KD. This claim
that the Michaelis constant reflects NAD+ binding is supported
by the similar Km and KD values obtained in dilute solution
(Table 1). Furthermore, the binding curve obtained in glucose
from the fluorescence experiment yielded a relative KD value
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addition to excluded volume, must be considered.33,60,66 Soft
interactions have begun to be characterized in the context of
protein binding and stability,62−65 but there is still a critical
need to understand these interactions in the context of enzyme
kinetics.
that is within error of the relative NAD+ Km value, both of
which were close to 1 (Figure 5). Thus, the negligible change in
Km with the addition of glucose or dextran (Figure 2B) for the
isopropanol oxidation can be ascribed to the sugars having little
to no effect on YADH−NAD+ binding. Because the Michaelis
constant does appear to approximate the KD value for
isopropanol oxidation, the fact that the relative Km values for
glucose and dextran are similar suggests that macromolecular
crowding has little effect on the binding of YADH to NAD+.
Crowder Size. At first glance, our observation that the effect
of dextran on YADH kinetics is independent of polymer size
contradicts previous trends with enzymes of similar dimensions.15,24,31 An extensive study by the Mas group reported
larger decreases in both Vmax and Km values with larger dextran
polymers, revealing that obstacle size plays a major role in the
magnitude of crowding effects for >100 kDa enzymes.32 The
authors explain that large crowders reduce the frequency of
enzyme−substrate encounters, but this decrease is partially
counterbalanced by caging effects with smaller crowders. Unlike
the enzymes used in the Mas study, our current and previous
data23 suggest that YADH is not limited by substrate−enzyme
encounters, even under crowded conditions.
Our previous work with malate dehydrogenase (MDH) also
reveals a nontrivial dextran size dependence for kinetic
crowding effects,29 even though this enzyme shares the same
rate-limiting step as YADH, the release of NADH. Importantly,
the decrease in Vmax in the presence of glucose is similar for
both MDH and YADH (∼50%), further confirming our theory
that the presence of this small molecule, which cannot exclude
volume, likely impedes product release. In addition to slowing
diffusion, the presence of dextran introduces an additional
excluded volume effect on MDH that further decreases the
Vmax. Such crowding effects are usually size-dependent. In fact,
in the presence of dextrans comparable in size to MDH (70
kDa), the relative Vmax was 40% lower than in the presence of
glucose. In contrast, while the YADH relative Vmax values in the
presence of dextran are slightly greater than for glucose, the
values never differ by >15%. Thus, the reason the effects of
dextran on YADH kinetics are size independent is because our
results indicate that excluded volume plays only a minor role.
Rather, as evidenced by the similar effects of dextran and
glucose, our work reveals that factors other than excluded
volume may be at play. This finding is consistent with a recent
computational study revealing that excluded volume is less
important than typically assumed.33 Similarly, a surge of recent
reports have emphasized the importance of considering soft
interactions and the chemical effects introduced by crowding.60−65 Thus, it is not surprising that these trends observed
with YADH would be independent of dextran size.
■
AUTHOR INFORMATION
Corresponding Author
*Address: 300 Pulteney St., Geneva, NY 14456. E-mail: slade@
hws.edu. Phone: (315) 781-3613. Fax: (315) 781-3860.
Funding
This work was supported by Single-Investigator Cottrell
College Science Award 20963 from Research Corporation for
Science Advancement and by the Hobart and William Smith
Provost office.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Doug Fuchs for generating the graphic for the table
of contents and Bradley Cosentino for his consultation on the
statistical analysis. We also appreciate the insightful discussions
about and helpful comments on the manuscript of David Slade,
Samuel Schneider, and Gary Pielak.
■
ABBREVIATIONS
NAD+ and NADH, oxidized and reduced β-nicotinamide
adenine dinucleotide, respectively; YADH, yeast alcohol
dehydrogenase; LDH, lactate dehydrogenase; MDH, malate
dehydrogenase; BSA, bovine serum albumin; Km, Michaelis
constant; Vmax, maximal rate under Michaelis−Menten kinetics.
■
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CONCLUSION
The results presented here directly highlight the critical role
that enzyme mechanism plays in macromolecular crowding
effects. For a single enzyme, dextran was able to increase or
decrease Vmax values depending on the rate-limiting step of the
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